STUDY QUESTION Do testis-specific cells have a normal karyotype in non-mosaic postpubertal Klinefelter syndrome (KS) patients with focal spermatogenesis and in non-mosaic prepubertal KS boys? SUMMARY ANSWER Spermatogonia have a 46, XY karyotype, and Sertoli cells surrounding these spermatogonia in postpubertal patients also have a 46, XY karyotype, whereas, in prepubertal KS boys, Sertoli cells surrounding the spermatogonia still have a 47, XXY karyotype. WHAT IS KNOWN ALREADY A significant proportion of patients with non-mosaic KS can have children by using assisted reproductive techniques thanks to focal spermatogenesis. However, the karyotype of the cells that are able to support focal spermatogenesis has not been revealed. STUDY DESIGN, SIZE, DURATION Testicular biopsy samples from non-mosaic KS patients were included in the study. Karyotyping for sex chromosomes in testis-specific cells was performed by immunohistochemical analysis of inactive X (Xi) chromosome and/or fluorescent in situ hybridization (FISH) analysis of chromosomes 18, X, and Y. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 22 KS patients (17 postpubertal and 5 prepubertal) who were non-mosaic according to lymphocyte karyotype analysis, were included in the study. After tissue processing, paraffin embedding, and sectioning, the following primary antibodies were used for cell-specific analysis and Xi detection; one section was stained with MAGE A4 for spermatogonia, SOX9 for Sertoli cells, and H3K27me3 for Xi; the other one was stained with CYP17A1 for Leydig cells, ACTA2 for peritubular myoid cells, and H3K27me3 for Xi. Xi negative (Xi−) somatic cells (i.e. Sertoli cells, Leydig cells, and peritubular myoid cells) were evaluated as having the 46, XY karyotype; Xi positive (Xi+) somatic cells were evaluated as having the 47, XXY. FISH stain for chromosomes 18, X, and Y was performed on the same sections to investigate the karyotype of spermatogonia and to validate the immunohistochemistry results for somatic cells. MAIN RESULTS AND THE ROLE OF CHANCE According to our data, all spermatogonia in both postpubertal and prepubertal non-mosaic KS patients seem to have 46, XY karyotype. However, while the Sertoli cells surrounding spermatogonia in postpubertal samples also had a 46, XY karyotype, the Sertoli cells surrounding spermatogonia in prepubertal samples had a 47, XXY karyotype. In addition, while the Sertoli cells in some of the Sertoli cell-only tubules had 46, XY karyotype, the Sertoli cells in some of the other Sertoli cell-only tubules had 47, XXY karyotype in postpubertal samples. In contrast to the postpubertal samples, Sertoli cells in all tubules in the prepubertal samples had the 47, XXY karyotype. Our data also suggest that germ cells lose the extra X chromosome during embryonic, fetal, or neonatal life, while Sertoli cells lose it around puberty. Peritubular myoid cells and Leydig cells may also be mosaic in both postpubertal patients and prepubertal boys, but it requires further investigation. LIMITATIONS, REASONS FOR CAUTION The number of prepubertal testicle samples containing spermatogonia is limited, so more samples are needed for a definitive conclusion. The fact that not all the cell nuclei coincide with the section plane limits the accurate detection of X chromosomes by immunohistochemistry and FISH in some cells. To overcome this limitation, X chromosome analysis could be performed by different techniques on intact cells isolated from fresh tissue. Additionally, there is no evidence that X chromosome inactivation reoccurs after activation of the Xi during germ cell migration during embryogenesis, limiting the prediction of X chromosome content in germ cells by H3K27me3. WIDER IMPLICATIONS OF THE FINDINGS Our findings will lay the groundwork for new clinically important studies on exactly when and by which mechanism an extra X chromosome is lost in spermatogonia and Sertoli cells. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by The Scientific and Technological Research Council of Türkiye (TUBITAK) (2219 – International Postdoctoral Research Fellowship Program for Turkish Citizens) and the Strategic Research Program (SRP89) from the Vrije Universiteit Brussel. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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具有局灶性精子发生的非嵌合型青春期后 Klinefelter 患者和非嵌合型青春期前 Klinefelter 男孩的睾丸嵌合体


研究问题：在具有局灶性精子发生的非嵌合型青春期后 Klinefelter 综合征 （KS） 患者和非嵌合型青春期前 KS 男孩中，睾丸特异性细胞是否具有正常的核型？总结答案精原细胞具有 46， XY 核型，青春期后患者围绕这些精原细胞的支持细胞也具有 46， XY 核型，而在青春期前 KS 男孩中，精原细胞周围的支持细胞仍然具有 47， XXY 核型。已知的是，由于局灶性精子发生，很大一部分非嵌合型 KS 患者可以通过使用辅助生殖技术生育孩子。然而，能够支持局灶性精子发生的细胞的核型尚未揭示。研究设计、规模、持续时间 来自非嵌合型 KS 患者的睾丸活检样本被纳入研究。通过对无活性的 X （习） 染色体进行免疫组织化学分析和/或对 18 、 X 和 Y 染色体进行荧光原位杂交 （FISH） 分析，对睾丸特异性细胞中的性染色体进行核型分析。经过组织处理、石蜡包埋和切片后，以下一抗用于细胞特异性分析和 习 检测;切片用 MAGE A4 染色精原细胞，SOX9 染色支持细胞，H3K27me3 染色 习;另一个用 CYP17A1 染色 Leydig 细胞，ACTA2 染色肾小管周围肌样细胞，H3K27me3 染色 习。习 阴性 （习−） 体细胞（即 支持细胞、睾丸间质细胞和肾小管周围肌样细胞）被评估为具有 46，XY 核型;习 阳性 （习+） 体细胞评估为具有 47， XXY。对同一切片进行 18 、 X 和 Y 染色体的 FISH 染色，以研究精原细胞的核型并验证体细胞的免疫组化结果。主要结果和机会的作用 根据我们的数据，青春期后和青春期前非嵌合型 KS 患者的所有精原细胞似乎都有 46 个 XY 核型。然而，虽然青春期后样本中精原细胞周围的支持细胞也具有 46， XY 核型，但青春期前样本中精原细胞周围的支持细胞具有 47， XXY 核型。此外，虽然一些仅支持细胞小管中的支持细胞在青春期后样本中有 46， XY 核型，但其他一些仅支持细胞小管中的支持细胞在青春期后样本中有 47， XXY 核型。与青春期后样本相比，青春期前样本中所有小管中的支持细胞均具有 47，XXY 核型。我们的数据还表明，生殖细胞在胚胎、胎儿或新生儿期间会丢失额外的 X 染色体，而支持细胞会在青春期左右丢失。小管周围肌样细胞和 Leydig 细胞也可能在青春期后患者和青春期前男孩中呈镶嵌状，但需要进一步研究。局限性，谨慎原因 含有精原细胞的青春期前睾丸样本数量有限，因此需要更多的样本才能得出明确的结论。并非所有细胞核都与截面平面重合这一事实限制了免疫组化和 FISH 在某些细胞中对 X 染色体的准确检测。 为了克服这一限制，可以通过不同的技术对从新鲜组织中分离的完整细胞进行 X 染色体分析。此外，没有证据表明 X 染色体失活在胚胎发生过程中生殖细胞迁移过程中 习 激活后再次发生，从而限制了 H3K27me3 对生殖细胞中 X 染色体含量的预测。研究结果的更广泛影响我们的研究结果将为新的临床重要研究奠定基础，这些研究究竟何时以及通过何种机制在精原细胞和支持细胞中丢失额外的 X 染色体。研究资金/竞争利益 本研究由土耳其科学技术研究委员会 （TUBITAK）（2219 – 土耳其公民国际博士后研究奖学金计划）和布鲁塞尔自由大学的战略研究计划 （SRP89） 资助。作者声明没有利益冲突。试验注册号 N/A。